Regulatory Effect And Mechanism Of NO On Germination And Cd Accumulation Of Alfalfa (Medicago Sativa L.) Seeds Under Cd Stress

Cadmium?Cd?is one of the most hazardous and ubiquitous contaminants.Nitric oxide?NO?,a common signal molecule in plant growth regulation biology,it has been reported to participate in all kinds of plant responses to Cd stress.Alfalfa?Medicago sativa L.?,capable of uptaking high concentration of Cd,Cu and Zn from soils and accumulate in bodies,is a promising plant for phytoremediation.In the entire vegetative cycle,seed germination is the most exuberant stage in crops for physiological metabolism.In this study,Cd stress was simulated by CdCl₂,while alfalfa seedlings were separately treated with a NO synthase inhibitor L-NAME?N'-nitro-L-arginine-methyl ester hydrochloride?,a nitrate reductase inhibitor NaN₃,a NO donor SNP?Na₂[Fe?CN?₅NO]?2H₂O?and a NO scavenger c-PTIO?2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide?to investigate the pathway of NO generation in alfalfa seedlings under Cd stress.The results were also used to discuss the seed germination,storage substances reserve,membrane lipid peroxidation,antioxidant enzymes activities of alfalfa under Cd stress.Knowledge of physiological and ecological effects of Cd stress to alfalfa during germination will help find ways to improve the seed germination rate and provide scientific basis for remediation of Cd contamination.The results showed as follows:?1?Cd stress increased the endogenous NO of alfalfa during germination,which indicated NO signal that was related to Cd resistance in seeds.In the experimental process from 1st day to 5th day,the resultant of NO content had significant changes;in terms of NO content in alfalfa with Cd treatments were significantly higher than that in control.With the increasing concentration of Cd,NO content in some samples experienced a rising tendency,and in the rest of samples had an increasing trend,followed by a decrease of NO content.It revealed that endogenous NO produced in alfalfa seeds under Cd stress,and 50?mol/L Cd has the most effective effect to NO content.NOS,NR,NO₂^-,Car were selected as the representative of path of NO produced.The results showed that,along with increment of Cd processing concentration,1st day and 3rd day and the 4th day of NOS activity presented the significant dose effect,namely under the low concentration the activity was strengthened,while under the high potency it was weakened;NOS activity of 2nd day and 5th day generally exhibited a gradual increase tendency.On the other hand,addition of Cd caused a significant change in NO₂^-and Car content in alfalfa seeds during germination.With the increasing concentration of Cd,NO₂^-and Car content in some samples NOS exhibited an increasing trend followed by a decrease of NO₂^-and Car content.The four parameters above were similar to the NO changing rule in general,which revealed that the production of endogenous NO could have been induced by Cd stress during the germination of alfalfa seed mainly through NOS catalytic,NO₂^-in NR catalysis or under the mediating role of Car and light.With the increasing concentration of Cd,the resultant NR activity in some samples experienced a more stable tendency in Car content,NOS and NO₂^-activities.It demonstrates that there is no significant relation existing between NR activity and Cd concentration during germination of alfalfa seeds.Significant downward tendency in NO content and in NOS,NR and NO₂^-activities were noticeably observed on the 1st day and 2nd day of treatment while from 3rd day to 5th day of treatment we noticeably observed increase in NO content;similar results were found in NOS,Car and NO₂^-.This indicates that seed germination might be mediated by NOS,NR,NO₂^-during the early stage of seed germination,with NOS,Car,NO₂^-playing the main role during the late days.In all,the addition of 50?mol/L Cd had the most significant alleviating effect against Cd stress for in this concentration,with NOS,NR activities and NO₂^-,Car content rising significantly,as well as the highest increase of NO content.?2?Cd stress significantly inhibited the germination and growth of alfalfa seeds,in terms of germination rate,germination energy,seed vigor index and germination index,the length of bud and hypocotyls,fresh weight of bud and hypocotyls.Addition of SNP was revealed to alleviate the negative effects of Cd on germination and seedling growth.Effects of SNP could be suppressed by NO scavenger c-PTIO,thus the production of endogenous NO induced by Cd stress during the germination of alfalfa seed,and both endogenous and exogenous NO were involved in the process of alfalfa seeds response to Cd stress by increasing germination rate,germination energy,seedling vigor index and germination index,the length of bud and hypocotyls,fresh weight of bud and hypocotyls.NOS and NR play important role in plants by producing NO under adversity stress.In this study,compared with treatment of Cd,the supply of L-NAME,NaN₃,NaN₃+L-NAME significantly decreased the length of bud and hypocotyls under Cd stress.The length of bud and hypocotyls with L-NAME treatments were significantly higher than that with NaN₃.Germination rate,germination energy,seed vigor index and germination index,fresh weight and dry weight of bud had the similar trend.It showed that NaN₃,L-NAME significantly inhibited the germination and growth of alfalfa seeds by inhibiting the activity of NR,NOS,further it was revealed that NR and NOS promoted the production of endogenous NO during the germination of alfalfa seed;increased the seed germination rate,germination energy,seed vigor index and germination index,the length of bud and hypocotyls,fresh weight and dry weight of bud and hypocotyls;alleviated the negative impact of Cd stress on germination and seedling growth.On the other hand,NR played a major role than NOS in terms of the promotion of germination and growth of alfalfa seeds.?3?Germination process is characterized by reserves mobilization such as carbohydrates and proteins where the newly formed products are used in formation of new structures.These reserve materials are made available by hydrolytic enzymes to deliver fuel for respiration and various anabolic reactions in the form of anabolites.Enzymes accountable for starch breakdown and mobilization of these reserves materials are ?-amylases and ?-amylases,and Cd has been reported to cause a restriction in these reserve mobilization.In this study,the content of reducing sugar,soluble sugar,soluble protein,crude fat and activity of ?-amylase,?-amylase,proteinase,and lipase were significantly inhibited under Cd stress on the other hand they were stimulated by SNP while the effects of SNP could be inhibited by c-PTIO.This shows that Cd stress induced the production of endogenous NO which had a regulating effect on seed storage material,in terms of increased the content of reducing sugar,soluble sugar,soluble protein,crude fat and activity of ?-amylase,?-amylase,proteinase,lipase in response to Cd stress in alfalfa seeds.Compared with Cd treatment alone,the supply of L-NAME,Na N3,NaN₃+L-NAME significantly decreased the content of soluble protein,crude fat and activity of ?-amylase,?-amylase,proteinase,lipase under Cd stress.Similarly,compared with CK,L-NAME,NaN₃,NaN₃+L-NAME treatments were significantly inhibited the content of reducing sugar,soluble protein,crude fat and activity of ?-amylase,?-amylase,proteinase,lipase.It showed that NaN₃,L-NAME significantly inhibited the storage material of alfalfa seeds by inhibiting the activity of NR,NOS,which showed that that endogenous NO alleviated the negative effects of Cd on storage material of alfalfa seeds.?4?As an active redox metal,Cd is able to induce the overproduction of ROS such as O₂^-and H₂O₂ produced in the seedlings under various abiotic stresses which in turn lead to membrane peroxidation;to cope and neutralize the toxicity of ROS,plants have evolved endogenous systems of anti-oxidative enzyme system such as CAT,POD,SOD,APX,GR,etc.In this study,the content of MDA,H₂O₂,O₂^-·,REC with Cd treatment were significantly higher than that in CK.The increase in activity of SOD,CAT,POD,APX,LOX resulted in alleviation of membrane lipid peroxidation,thus and so improve the resistance to Cd stress.While compared with Cd treatment,supply of SNP significantly reduced the content of MDA,H₂O₂,O₂^-·,REC and activity of SOD,CAT,APX,LOX in the seeds under Cd stress,showed that ROS free radicals increased and membrane lipid peroxidation which took place in cell membrane system under Cd stress;and the addition of exogenous NO alleviated the membrane lipid peroxidation.Alleviation of SNP could be suppressed by c-PTIO with a significant increase in the above parameters than Cd+ SNP.It revealed that the removal of NO caused the increase in content of ROS free radicals level and stimulated the antioxidant system of alfalfa seeds.Under Cd stress,supply of NaN₃,L-NAME,NaN₃+L-NAME significantly increased the content of MDA,H₂O₂,O₂^-·,REC and the activity of SOD,CAT,APX,LOX more than Cd did,this means that NaN₃,L-NAME significantly increased the content of ROS free radicals level in alfalfa seeds by promoting the activity of NR,NOS;further NO inhibit the plant from oxidation damage by regulating general mechanism for cellular redox homeostasis and promoting the transformation of O₂^-· to H₂O₂ and O₂,also by enhancing H2O2-scavenging enzyme activities.In general,the content of MDA,H₂O₂,O₂^-·,REC,Pro as well as the activity of SOD,CAT,APX,GR showed a gradual increase trend over time.This means that in time-dependent manner Cd induced the over-accumulation of these molecules leading to the oxidative injury in alfalfa seeds and inhibiting seedling growth.On the other hand,POD,LOX had a decreasing trend followed by an increase in activity,which means that the relation between NO-induced,POD and LOX activities is complex and still needs further researches.?5?Cd content was extremely lower in alfalfa seeds sown in CK conditions,however SNP,L-NAME,NaN₃,L-NAME+NaN₃,c-PTIO treatments resulted in a significant increase in Cd level which can be suggested as a "false positive" effect,the reason why further researches are recommended.Under Cd stress,apply of SNP significantly increased the Cd content than in only Cd-treated sample with a maximum accumulation of 24.945?g/g.Effects of SNP could be suppressed by c-PTIO,which significantly decreased the Cd content than Cd +SNP.It illustrated that SNP promoted the germination and growth of plant,as well as the up-taking of Cd.However,it differed with the conclusion of predecessors in alfalfa seedlings.It suggested that a part of Cd forms complexes which are not toxic,thus not caused fatal damage to seed germination.Under Cd stress,Cd accumulation with the supplies of L-NAME,NaN₃,L-NAME+NaN₃ were significantly lower than Cd treatment,which showed the removal of NO and the inhibition of NO enzymes caused the block of Cd accumulation of alfalfa seeds.It illustrated that endogenous NO was involved in the process of alfalfa seeds response to Cd stress and improved the absorption and accumulation of alfalfa seeds to Cd.To sum up,the present work demonstrates that Cd stress induces the production of endogenous NO,which has significant effect on seed germination and seedling growth,storage materials,ROS metabolism and Cd accumulation in alfalfa;and their regulation is promoted by addition of exogenous NO,hence reversing the negative impact of Cd on germination.Inhibition of NOS,NR activity and removal of NO will offset the positive role of SNP treatment.The effective concentration of Cd that promotes endogenous NO generated in this study is 50?mol/L.Alfalfa plays important role in remediation of heavy metals like Cd,Cu,Pb etc.contaminated soils.In China,a large plantation area of alfalfa constitutes the basis in the development of animal husbandly.Photosynthesis as a basis of physiological metabolism development,effects of NO on photosynthetic rate,transpiration rate,stomatal conductance,intercellular CO₂ concentration,total chlorophyll content and xanthophyll cycle in alfalfa under Cd stress should be investigated in future researches.Further researches are recommended to elucidate genetic and proteomic analyses and additional physiological approaches to understand the details of exogenous NO in metabolic functions in plant,furthermore it will be helpful for elucidating involved mechanisms for better application in phytoremediation of heavy metals contaminated soils.